Process for sequence saturation mutagenesis (sesam)

ABSTRACT

A process for the mutagenesis of a double-stranded polynucleotide sequence (master sequence) of n base-pairs having a (+)-strand and a complementary (−)-strand comprising the steps (i) creation of a collection of single-stranded fragments of the (+)-strand of the master sequence wherein all members of the collection have the same 5′-terminus and have a deletion in the 3-terminus such that the collection represents (+)-strands with a length of n−1, n−2, n−3, . . . nucleotides; (ii) introduction of at least one universal or degenerate nucleotide at the 3′-terminus of the (+) strand produced in step (i); (iii) elongation of the (+)-strand produced in step (ii) to the full length of the master sequence using the (−)-strand or fragments thereof as a template strand for the elongation; (iv) synthesis of a (−)-strand by using the (+)-strand produced in step (iii) as a template strand thereby effecting mutations in the (−)-strand at the positions of the previous universal or degenerate nucleotides compared to the master sequence.

Inspired by Darwinian evolution in nature, random mutagenesis methodshave been developed for tailoring proteins to our needs and elucidatingstructure-function relationships (1). Creating diversity on the geneticlevel is the complementary tool to gene shuffling since it creates theopportunity to introduce novel mutations for adapting proteins tonon-natural environments in biotechnological processes. The sequencespace of a truly randomized library is, by its nature, not limited by apre-selection for function under physiological conditions. Parentalgenes used in gene shuffling experiments are optimized by naturalevolution for function under physiological conditions.

Among random mutagenesis methods, error-prone PCR methods based oninaccurate amplification of genes are most commonly used due to theirsimplicity and versatility. Error-prone PCR methods can be divided intothree categories: A) Methods that reduce the fidelity of the polymeraseby unbalancing nucleotides concentration and/or adding of manganesechloride (2-4), B) Methods that employ nucleotide analogs (5,6), and C)Combined methods (A and B; (7)).

SPECIFICATION

The invention relates to a process for the mutagenesis of adouble-stranded polynucleotide sequence (master sequence) of nbase-bairs having a (+)-strand and a complementary (−)-strand comprisingthe steps

-   (i) creation of a collection of single-stranded fragments of the    (+)-strand of the master sequence wherein all members of the    collection have the same 5′-terminus and have a deletion in the    3′-terminus such that the collection represents (+)-strands with a    length of n−1, n−2, n−3, . . . nucleotides;-   (ii) introduction of at least one universal nucleotide or degenerate    nucleotide at the 3′-terminus of the (+) strand produced in step    (i);-   (iii) elongation of the (+)-strand produced in step (ii) to the full    length of the master sequence using the (−)-strand or fragments    thereof as a template strand for the elongation;-   (iv) synthesis of a (−)-strand by using the (+)-strand produced in    step (iii) as a template strand thereby effecting mutations in the    (−)-strand at the positions of the previous universal nucleotides or    degenerate nucleotides compared to the master sequence.

A universal nucleotide is a nucleotide that can pair to all fourstandard nucleotides. For example deoxyinosinetriphosphate (dITP) whenincorporated into a polynucleotide strand allows base pairing withAdenine, Guanine, Thymine and Cytosine.

Preferred universal nucleotides are deoxyinosine, 3-nitropyrrole and5-ntroindole.

A degenerate nucleotide is a nucleotide that can pair to less than allfour standard nucleotides. Preferred degenerate nucleotides areN⁶-methoxy-2,6-diaminopurine (K), N⁶-methoxy-aminopurine (Z),hydroxylaminopurine (HAP), 2′-deoxyribonucleoside triphosphate (dyTP),6H,8H-3,4-dihydropyrimidol [4,5-c][1,2]oxazin-7-one (P),N⁴-aminocytidine, N⁴-hydroxy-2′-deoxycytidine,N⁴-methoxy-2′-deoxycytidine and 8-oxodeoxyguanosine triphosphate(8-oxo-G).

A nucleotide analog with promiscuous base pairing property means that anucleotide that is based on a purine or pyrimidine structure (e.g. A, G,C, T) which is modified in one or more of its functional groups, canhave base pairs to more than one other nucleotides, e.g. to two, threeor four nucleotides. Also the universal nucleotides and degeneratenucleotides are embraced by the term nucleotide analog with promiscuousbase pairing property.

A preferred embodiment of the invention is a process as described above,wherein an oligonucleotide of the general formulap(U)_(a)(N)_(b)*(s)_(c)[TERM]

with

-   -   p=5′-phosphate or hydroxy-group or any chemical group capable of        forming diester bonds    -   U=universal or degenerate bases    -   a=arbitrary integral number from 0 to 10000, preferred from        1-100    -   N=mixture of four bases (A/T/G/C (standard nucleotides)    -   b=arbitrary integral number from 0 to 100, preferred from 1-10,    -   *=cleavable group such as phosphothioate bonds in phosphothioate        nucleotides    -   S=standard nucleotide or nucleotide analog    -   c=arbitrary integral number from 0 to 100, preferred from 1-10,    -   [TERM]=a dye terminator or any group preventing elongation of        the oligonucleotide, with the proviso that a+b>0, preferred >1,        more preferred >2, is used in step (ii) to introduce universal        or degenerate bases to the collection of single-stranded        fragments created in step (i).

[TERM] can be any group which prevents the elongation of theabove-mentioned oligonucleotide, preferably an hydrogen or a dyeterminator. Preferred dye terminators are Coumarin, 6-FAM, Fluorescein,Fluorescein-dT, JOE, Oregon Green, ROX, TAMRA or Texas Red-X.

Another preferred embodiment of the invention is the creation of acollection of single-stranded fragments of the (+)-strand of the mastersequence according to step (i) by incorporating alpha-phosphothioatenucleotides, preferably dATPαS, dGTPαS, dTTPαS, dCTPαS, into the PCRproducts and subsequent cleavage of the phosphothioate bond by iodineunder alkaline conditions.

Another preferred embodiment of the process according to the inventionis the following process for step (iii): Starting from a double-strandedplasmid which harbors the master sequence a (−) single stranded plasmidpolynucleotide sequence is synthesized using a primer which annealsdownstream of the (+)-strand of the master sequence. This (−) singlestranded plasmid polynucleotide sequence is annealed with the(+)-strands produced in step (ii). The (+)-strands are elongated to thefull length of the master sequence using the (−)-strand as a template.This process is shown in FIG. 3.

Further preferred embodiments are disclosed in the claims.

Step (i): Creating DNA Fragment Pool with Length Distribution

In the first step, PCR is performed using biotinylated forward primerand non-biotinylated reverse primer in the presence of both standardnucleotides and α-phosphothioate nucleotides. α-Phosphothioatenucleotides are similar to normal nucleotides, except that an oxygenatom of α-phosphate is replaced by a sulphur atom. Phosphothioate bondis susceptible to iodine cleavage in alkaline condition. Due to randomdistribution of phosphothioates in the DNA, a library of fragments thatstop at every single base is generated in a single PCR. Nicks may formif the binding between two complementary strands is strong. Biotinylatedfragments are isolated using Strepavidin-coated biomagnetic beads. DNAmelting solution (0.1 M NaOH) is then used to remove non-biotinylatedstrands and undesired fragments. Biotinylated fragments can easily bereleased from biomagnetic beads by boiling in 0.1% SDS solution. Ascheme of the first step is shown in FIG. 1 a and the correspondingexperimental data is shown in FIG. 1 b.

Step (ii): Enzymatic Elongation

To elongate DNA fragments with universal bases or degenerate bases (FIG.2), two approaches can be used. In a first approach terminaldeoxynucleotidyl transferase has been used for incorporating ambiguousbases (universal base or degenerate base) at the 3′-termini. The secondapproach requires single-stranded DNA ligation between DNA fragments anda “special” oligo. This “special” oligo is 5′-phosphorylated tofacilitate ligation. It is terminated with fluorescein to avoid intra-and intermolecular ligation and to quantify the incorporation. There are3 distinct parts in this oligo: 1) “Mutational part” containinguniversal bases or degenerate bases, 2) “Adhesive part” consisting ofthree bases that encompasses all 64 possibilities by using equimolar ofA/T/G/C in oligo synthesis. This part is designed to assist annealing inthe subsequent PCR used for the full length gene synthesis. The“Redundant part” is connected to “Adhesive part” via phosphothioate bondthat allows its cleavage by the iodine method. ssDNA ligation can beaccomplished using ThermoPhage RNA Ligase II (Prokaria). ThermoPhage RNALigase 11 catalyses the ATP-dependent intra- and intermolecularformation of phosphodiester bonds between 5′-phopsphate and 3′-hydroxyltermini of single-stranded DNA or RNA. This enzyme is derived fromthermophilic phage TS2126 that infects the thermophilic eubacteriumThermus scotoductus. This thermostable enzyme is homologous to RNAligase derived from bacteriophage T4. It shows superior efficiency inssDNA ligation as compared to T4 RNA ligase. The ligation efficiency wasdetermined by ligating a fluorescein labelled oligo to single strandedDNA template. After the ligation, the ‘Redundant part’ is removed byiodine cleavage in alkaline condition.

Step (iii): Full-Length Gene Synthesis

The third step is extending the elongated fragments to full-length (FIG.3). Here, we use a single-stranded template to avoid the wild-typeamplification. Single-stranded template is synthesized using reverseprimer. Methylated and hemimethylated parental genes are removed by DpnI digestion. This procedure is similar to QuikChange Site-DirectedMutagenesis (Stratagene) except that only one non-mutagenenic primer isused instead of a pair of mutagenic primers. Elongated fragments annealto single-stranded template due to complementarities and extend thesingle strand to full-length. Reverse primers present in this PCRreaction can only anneal to the newly synthesized full-length singlestrand and not to single-stranded template. After reverse primer bindingthe double stranded full-length genes will be synthesized.Double-stranded DNAs will contain nucleotide analogs in one strand andstandard nucleotides in another strand.

In the event where a double-stranded template is used in this PCRinstead of a single-stranded one we would observe that the reverseprimer binds to its complementary template strand, amplifying doublestranded DNA that do not contain nucleotide analogs.

Step (iv): Nucleotide Replacement

In the last PCR the nucleotide analog-containing strands are used astemplates to replace nucleotide analogs with standard nucleotides (FIG.4). After restrictive digestion, mutated genes are cloned into suitableexpression vector, transformed and expressed in E. coli. Randomlyselected clones were picked and grown in small cultivation tubes (5 ml;LB_(amp)). Isolated plasmid DNA is subsequently sequenced. Preliminarysequencing results with 100 clones proved the proper replacement andshowed a bias as expected for inosine (FIG. 5 and FIG. 6).

Generation of mutant libraries using the SeSaM method can be completedwithin 1-2 days. The SeSaM method offers the following advantages:

-   1) Able to saturate every single position of a sequence with all 20    possible naturally occurring amino acids.-   2) No bias in mutational spectra if truly universal bases are used.-   3) Mutation spectra can be manipulated using transition-favoured or    transversion-favoured degenerate bases.-   4) Controllable length of mutation regions by designing proper    special oligos.-   5) Fragment size distribution can be controlled by using    Sp-dATPαS/Sp-dTTPαS/Sp-dGTPαS/Sp-dCTPαS or combination of them.    Materials and Methods

All chemicals used were of analytical-reagent grade or higher qualityand were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen,Germany), Applichem GmbH (Darmstadt, Germany) or Carl Roth GmbH+Co(Karlsruhe, Germany). pEGFP plasmid was purchased from BD Biosciences(Heidelberg, Germany).

A thermocycler (Mastercycler gradient; Eppendorf, Hamburg, Germany) andthin-wall PCR tubes (Mμlti-Ultra tubes; 0.2 ml; Carl Roth GmbH+Co.,Karlsruhe, Germany) were used in all PCRs. The reaction volume of allPCRs was always 50 μl.

(Preparatory Step 1) Single-Stranded pEGFP Preparation:

For each PCR, 5 U Pfu Turbo polymerase (Stratagene, Amsterdam,Netherlands), 0.2 mM dNTP mix (New England Biolab, Frankfurt, Germany),12.6 μmol reverse primer (5′-GACCGGCGCTCAGTTGGMTTCTAG-3′) and 48.6-54.3ng plasmid pEGFP (Miniprep, Qiagen, Hilden, Germany) were used. AfterPCR (95° C. for 30 sec 1 cycle, 95° C. for 30 sec/55° C. for 1 min/68°C. for 4 min 40 cycles), 40 U of Dpn I was added followed by incubationat 37° C. for 3 hours. Product recovery was done using a NucleoSpinExtract (Macherey-Nagel, Düren, Germany; elution volume of 35 μl for 200μl PCR product).

(Preparatory Step 2) Cloning Vector Preparation:

24.3-27.1 μg plasmid pEGFP (Miniprep; QIAGEN) was first digested with 30U EcoRI (New England Biolab) in a reaction mixture of 100 μl. Thereaction mixture was incubated at 37° C. for 3 hours. The linearizedplasmid was purified with NucleoSpin Extract (Machery-Nagel; Elutionvolume of 50 μl for 100 μl reaction mixture). 4.9-5.6 μg of linearizedplasmid pEGFP was subjected to second digestion with 20 U Age I (NewEngland Biolab) in a reaction volume of 50 μl. After incubation at 37°C. for 3 hours, the double digested plasmid was purified with NucleoSpinExtract (Macherey-Nagel; elution volume of 35 μl for 50 μl reactionmixture). Double digested pEGFP was used for subsequent cloning.

(Step 1) PCR with dATPαS:

For each PCR (94° C. for 3 min 1 cycle, 94° C. for 1 min/59.5° C. for 1min/72° C. for 75 sec 31 cycles, 72° C. for 10 min 1 cycle), 2.5 U TaqDNA polymerase (Qiagen), 0.2 mM dNTP mix (New England Biolab), 0.2 mMSp-dATPαS (Biolog Life Science Institute, Bremen, Germany), 12.6 pmol5′-biotinylated forward primer (5′-GACCATGATTACGCCAAGCTTGC-3′), 12.6pmol reverse primer (5′-GAC CGGCGCTCAGTTGGAATTCTAG-3′) and 242.9-271.4ng plasmid pEGFP (Miniprep; Qiagen) were used.

(Step 2) Iodine Cleavage of Thiophosphodiester Backbone:

The phosphothiate bond was cleaved with iodine (dissolved in ethanol;final concentration in the PCR tube 2 μM). The mixture was incubated atroom temperature for 1 hour.

(Step 3) Preparation of Single Stranded DNA Fragments with DifferentLength:

Biotinylated DNA fragments from the forward primer were isolated usingDynabeads MyOne Streptavidin (DYNAL Biotech, Oslo, Norway) at roomtemperature. 50 μl of biomagnetic beads (10 mg/ml) were washed twiceusing 100 μl 2× B&W buffer (10 mM Tris-HCl, pH 7.5; 1.0 mM EDTA, 2.0 MNaCl). Washed biomagnetic beads were resuspended in 100 μl 2× B&W bufferand 100 μl of cleaved PCR product was added. After incubating for 20min, the biotinylated DNA fragments were immobilized on the biomagneticbeads and the iodine was removed by washing with 100 μl 2× B&W buffer.Non-biotinylated DNA fragments were released after incubating in 100 μlDNA melting solution (0.1 M NaOH) at 37° C. for 10 min followed by beadwashing steps with 100 μl of DNA melting solution and 100 μl 1× B&Wbuffer. The washed Dynabeads were boiled in 60 μl 0.1% SDS for releasingthe DNA fragments from the solid support. The supernatant containing theDNA fragments was transferred to another tube immediately.

(Step 4) SDS Salt Removal from Eluted Single Stranded DNA Fragments:

Desalting was done using NucleoTrap kit (Macherey-Nagel, Duren,Germany). 400 μl of buffer NT2 was added to 100 μl of eluted DNAfollowed by 15 μl of NucleoTrap suspension.

The mixture was incubated at room temperature for 10 min and gentlyshaked every 2-3 min. The sample was then centrifuged at 10000 g for 30sec and after discarding carding the supernatant, the beads were washedwith 500 μl of buffer NT3. The latter step was repeated once. The pelletwas air-dried for 15 min at 37° C. to remove residual ethanol,resuspended in 55 μl Tris/HCl buffer (5 mM; pH 8.5) and incubated forDNA elution at 50° C. for 5 min for DNA elution. The suspension waspipetted into NucleoSpin Microfilter and centrifuged at 10000 g for 30sec to separate beads from DNA containing solution.

(Step 5) Enzymatic Elongation of DNA Fragments with Universal Base:

Total reaction volume for each elongation reaction was 50 μl. In eachreaction, 5 U terminal transferase (New England Biolabs), 0.25 mM CoCl₂,0.4 μM dITP (Amersham Biosciences Europe GmbH, Freiburg, Germany), and18 μl desalted DNA from step 4 were used. After incorporation ofuniversal bases in the elongation reaction (37° C. for 30 min and heatdeactivation of the transferase at 70° C. for 10 min), the product waspurified following the QIAquick Nucleotide Removal Kit (QIAGEN; elutionvolume of 25 μl for 50 μl reaction mixture) protocol.

(Step 6) Full-Length Gene Synthesis:

For each PCR (94° C. for 3 min 1 cycle, 94° C. for 1 min/59.5° C.+0.2°C. (0.2° C. increment for each cycle) for 1 min/72° C. for 3 min 30cycles, 72° C. for 10 min 1 cycle), 2.5 U Taq DNA polymerase (Qiagen),0.2 mM dNTP mix (New England Biolab), 13.3 μl elongated DNA fragment, 20pmol reverse primer (5′-GAC CGGCGCTCAGTTGGMTTCTAG-3′) and 0.66-0.76 μgsingle-stranded reverse template (preparatory step 1) were used. Aftersynthesizing the full-length gene a purification step was performedusing the NucleoSpin Extract (Macherey-Nagel; elution volume of 35 μlfor 150 μl PCR product).

(Step 7) Universal Base Replacement:

For each PCR (94° C. for 3 min 1 cycle, 94° C. for 1 min/52.7° C. for 1min/72° C. for 75 sec 30 cycles, 72° C. for 10 min 1 cycle), 2.5 U TaqDNA polymerase (Qiagen), 0.2 mM dNTP mix (New England Biolabs), 20 pmolforward primer (5′-GACCATGATTACGCCAAGCTTGC-3′), 20 pmol reverse primer(5′-GAC CGGCGCTCAGTTGGAATTCTAG-3′) and 2.5 μl full-length gene (step 6)were used. The PCR product was purified using NucleoSpin Extract(Macherey-Nagel; elution volume of 50 μl for 150 μl PCR product).

(Step 8) PCR Product Digestion:

After universal base replacement, 40 μl of purified PCR product (step 7)was digested with 30 U EcoRI (New England Biolab) for 3 hours at 37° C.20 U of AgeI (New England Biolab) was then added to the digest and themixture was incubated for additional 3 hours at 37° C. The digestedproduct was purified using NucleoSpin Extract (Macherey-Nagel; elutionvolume of 25 μl).

(Step 9) Ligation and Transformation:

The ligation of the digested PCR product (step 8) and pEGFP cloningvector (preparatory step 2) was performed at room temperature for 1 hourusing T4 DNA ligase (Roche, Mannheim, Germany) and transformed into E.coli XL2 Blue (Stratagene, Amsterdam, Netherlands) cells. Competentcells were prepared by resuspending the cell pellet of a 50 ml culture(OD₅₇₈ 0.4-0.5) in 2 ml TSS buffer (10 g PEG 6000; 5 ml DMSO; 0.6 gMgSO₄; 100 ml LB). 5 μl ligation mixture was added to 200 μl cellaliquot followed by incubation in ice for 20 min, heat-shock s at 42° C.for 45 sec and additional chilling in ice for 2 min. After adding 0.8 mlof LB, the culture was shaken at 37° C. and 170 rpm for 1 hour. Cellswere harvested by centrifugation at 3000 g, room temperature for 2 min.900 μl of supernatant was discarded and cells were gently resuspended inthe remaining 100 μl of supernatant. The cells were then plated onLB/Amp plate and incubated at 37° C. for overnight.

DESCRIPTION OF THE FIGURES

FIG. 1: a) Step (i): A random fragment size distribution is created b)Upper and middle gel picture: PCR product before (left lane) and after(right lane) iodine cleavage. Lower gel picture: DNA fragment sizedistribution after DNA melting and purification for differentconcentration of Sp-dATPαS (stated at the lower part of the lane).

FIG. 2: a) Step (ii): DNA fragments elongated with universal ordegenerate bases b) Lower left gel picture: PCR with primers elongatedwith different concentration of deoxyinosine at 3′-termini usingterminal deoxynucleotidyl transferase. Lower right gel picture: PCR withprimers elongated with different concentration of 5-nitroindole at3′-termini using terminal deoxynucleotidyl transferase.

FIG. 3: Step (iii): Synthesis of full-length genes containing nucleotideanalogs

FIG. 4: Step (iv): Nucleotide analogs are replaced by standardnucleotides.

FIG. 5: Sequencing result of 100 randomly picked clones.

FIG. 6: Random distribution of mutations of 100 sequenced clones.

LITERATURE

-   1. Arnold, F. H., Wintrode, P. L., Miyazaki, K. and    Gershenson, A. (2001) Trends Biochem. Sci., 26,100-106.-   2. Cadwell, R. C. and Joyce, G. F. (1994) PCR Meth. App., 2,    136-140.-   3. Lin-Goerke, J. L., Robbins, D. J. and Burczak, J. D. (1997)    Biotechniques, 23, 409-412.-   4. Cadwell, R. C. and Joyce, G. F. (1992) PCR Meth. Appl., 2, 28-33.-   5. Kuipers, O. P. (1996) Meth. Mol. Biol., 57, 351-356.-   6. Zaccolo, M., Williams, D. M., Brown, D. M. and    Gherardi, E. (1996) J. Mol. Biol., 255, 589-603.-   7. Xu, H., Petersen, E. I., Petersen, S. B. and    el-Gewely, M. R. (1999) Biotechniques, 27, 1102-1108.

1. A process for the mutagenesis of a double-stranded polynucleotidesequence (master sequence) of n base-pairs having a (+)-strand and acomplementary (−)-strand comprising the steps (i) creating a collectionof single-stranded fragments of the (+)-strand of the master sequencewherein all members of the collection have the same 5′-terminus and havea deletion in the 3′-terminus such that the collection represents(+)-strands with a length of n−1, n−2, n−3, . . . nucleotides; (ii)introducing at least one universal or degenerate nucleotide at the3′-terminus of the (+)-strands produced in step (i); (iii) elongatingthe (+)-strands produced in step (ii) to the full length of the mastersequence using the (−)-strand or fragments thereof as a template strandfor the elongation; (iv) synthesizing a (−)-strand by using the(+)-strand produced in step (iii) as a template strand thereby effectingmutations in the (−)-strand at the positions of the previous universalor degenerate nucleotides compared to the master sequence.
 2. A processaccording to claim 1, wherein the collection of single-strandedfragments in step (i) is created by incorporating nucleotide analogs andsubsequent cleavage in alkaline or acidic solution.
 3. A processaccording to claim 2, wherein the nucleotide analog is analpha-phosphothioate nucleotide and oxidative cleavage is achieved byiodine at the phosphothioate bonds.
 4. A process according to claim 1,wherein step (ii) comprises the elongation of the collection of singlestranded fragments produced in step (i) with universal base ordegenerate base by enzymatic or chemical methods.
 5. A process accordingto claim 4, wherein terminal deoxynucleotidyl transferase or DNApolymerases or DNA/RNA ligases are used for elongation.
 6. A processaccording to claim 1, wherein deoxyinosine, 3-nitropyrrole,5-nitroindole or a nucleotide analog with promiscuous base pairingproperty is used as a universal nucleotide in step (ii).
 7. A processaccording to claim 1, wherein N⁶-methoxy-2,6-diaminopurine (K), N6-methoxy-aminopurine (Z), hydroxylaminopurine (HAP),2′-deoxyribonucleoside triphosphate (dyTP), 6H,8H-3,4-dihydropyrimidol[4,5-c][1,2]oxazin-7-one (P), N⁴-aminocytidine,N⁴-hydroxy-2′-deoxycytidine, N⁴-methoxy-2′-deoxycytidine,8-oxodeoxyguanosine triphosphate (8-oxo-G) or a nucleotide analog withpromiscuous base pairing property is used as degenerate nucleotide instep (ii).
 8. A process according to claim 1, wherein an oligonucleotideof the general formulap(U)_(a)(N)_(b)*(S)_(c)[TERM]with p=5′-phosphate or hydroxy-group or anychemical group capable of forming diester bonds U=universal ordegenerate bases a=arbitrary integral number from 0 to 10000 N=mixtureof four bases (A/T/G/C (standard nucleotides)) b=arbitrary integralnumber from 0 to 100 *=cleavable group such as phosphothioate bonds inphosphothioate nucleotides S=standard nucleotide or nucleotide analogc=arbitrary integral number from 0 to 100 [TERM]=a dye terminator or anygroup preventing elongation of the oligonucleotide, with the provisothat a+b>0, is used in step (ii) to introduce universal or degeneratebases to the collection of single-stranded fragments created in step(i).
 9. A process according to claim 8, wherein the oligonucleotide isdesigned in a way that (a) stop codons and/or (b) amino acids whichdisrupt secondary structures, are avoided in the collection of themutagenized polynucleotide sequences.
 10. A process according to claim8, wherein the oligonucleotide is designed in a way that (a) transitionmutations or (b) transversion mutations, are effected in the collectionof the mutagenized polynucleotide sequences.
 11. A process according toclaim 8, wherein the single-stranded fragment created in step (i) whichis not ligated with the oligonucleotide is removed using exonuclease.12. A process according to claim 1, wherein the elongation in step (iii)is effected by a PCR reaction.
 13. A process according to claim 1,wherein step (iii) comprises the synthesis of a (−)-single strandedplasmid polynucleotide sequence from a double-stranded plasmid harboringthe master sequence using a primer which anneals downstream of the(+)-strand of the master sequence, and annealing of this (−)-ss-plasmidpolynucleotide sequence with the (+)-strand produced in step (ii), andelongation of the (+)-strand.
 14. A process according to claim 1,wherein step (iii) comprises the synthesis of a (−)-single-strandedplasmid harboring the master sequence using a primer which annealsdownstream of the (+)-strand of the master sequence in the presence ofuracil and standard nucleotides and after the elongation of the(+)-strand produced in step (ii), the uracil carrying(−)-single-stranded plasmid is digested with uracil glycosylase.
 15. Aprocess according to claim 1, wherein a PCR amplification is used afterstep (iii) in order to synthesize a (−)-strand complementary to the(+)-strand produced in step (iii), thereby effecting a double-strandedmaster sequence carrying mutations.